AlanFung:LabNotes/CTCF/2011-6-28

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Revision as of 18:41, 30 June 2011 by >Alan6017518 (→‎Results)
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N9-MDA Bisulfite Converted Sequencing Library Consturction

Objective

  • Work on sperm/ESCs control cell samples plus GM12878 gDNA
  • Sample List
  1. A1-100 sperm cells
  2. A2-100 sperm cells (repeat)
  3. B1-100 ESCs
  4. B2-100 ESCs (repeat)
  5. C1-600pg GM12878 gDNA
  6. C2-600pg GM12878 gDNA>Noi
  7. C3-600pg GM12878 gDNA>Noi
  8. D-1.2ng GM12878 gDNA
  9. E-NTC

Overview

  • Proteinase K Digestion
  • Bisulfite conversion
  • MDA
  • EtOH precipitation
  • Nextera Library Construction (Low input protocol)
  • Page Analysis
  • Sanger Sequencing

Protocol

Proteinase K Digestion

  • Preparation of Proteinase K
  • Add 260 µl (D5020) of Proteinase K Storage Buffer to the tube containing Proteinase K. Dissolve completely
  • Label tubes A1, A2, B1, B2
  1. Sperms: ~50cell/tube; 15 tubes
  2. ES cells: ~50cell/tube; 15 tubes
  • Thaw 4 tubes on ice, we will use 100cells per reaction
  • Spin down tubes and add 9ul nuclease free water and transfer cells to 0.2ml tubes
Content Volume per Reaction
M-Digestion Buffer (2X) 10
Sample 9
Proteinase K 1
Total 20
  • Incubate the samples at 50C for 20min

Bisulfite Conversion

  • Dilute GM12878 gDNA from 50ng/ul to 0.5ng/ul (1:100)
  • Setup two more 600pg GM12878 gDNA reactions for Noi to perform capture using the 330k probe set
0.5ng/ul GM12878 gDNA Volume Per Reaction Make up to 20ul with H2O
600pg 1.2 18.8
1.2ng 2.4 17.6
  • Add 130ul CT Conversion Reagent to all samples
  • Perform reaction in thermocycler
  1. Step1 98C, 8m
  2. Step2 64C, 3.5hr
  3. Step3 4C, storage for up to 20 hr
  • Proceed with Zymo column based method with improvement tweak protocol
  • Elute with 11ul EB Buffer
  • Transfer bisulfite converted samples to 0.2ml tubes for MDA

Heat Denature

  • 92C for 30 sec
  • Transfer to ice immediately

MDA

  • Prepare MDA Master Mix
Content Volume Per Reaction MM
10X RepliPhi Buffer 2 22
1mM N9 Primer 1 11
25mM dNTP 0.8 8.8
RepliPhi Phi29 (100U/ul) 1 11
Klenow Exo Minus (10U/ul) 0.5 5.5
H2O 4.7 51.7
Total 10 110
  • Add 10ul of MDA mastermix to each sample
Plate Layout Plate Layout Plate Layout
1 2
A A1-100 Sperm Cells Noi-600pg GM12878
B A2-100 Sperm Cells Noi-600pg GM12878
C B1-100ESCs Empty
D B2-100ESCs Empty
E C-600pg GM12878 Empty
F D-1.2ng GM12878 Empty
G E1-NTC Empty
H E2-NTC(MDA) Empty
Plate Layout Plate Layout Plate Layout

30C 60 min, 85C 3 min, 4C hold

Ethanol Precipitation

  • Add 2.5X 100% Ethanol
  • Add 0.1X 3M NaOAC
  • Add 1ul glycoblue
  • Store at -80C for 30min (CONTINUE ON 6/29)
  • Spin down at 10,000rpm at 4C for 15min
  • Remove all liquid
  • Add 500ul 4C chilled 75% ethanol
  • Spin at 10,000rpm at 4C for 5 min
  • Remove all liquid
  • Let dry in hood for 5 min
  • Add 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
  • Let sit at RT to dissolve the pellet

Nextera Library Construction (Low input protocol)

  • In a qPCR tube, mix
  • 3ul genomic DNA
  • 1ul 1:5 Nextera illumina compatible transposomes (10 μl Nextera enzyme, 20 μl 1XTE, 20 μl 100% glycerol)
  • 1ul 5X NExtera HMW buffer
  • Incubate at 55C for 5 min
  • Setup PCR Reaction with Klenow Exo- but without primers
Content Volume Per Reaction MM
Water 17.5 154
DNA 5 Do not add to MM
KAPA Supermix 25 220
Klenow Exo Minus 0.5 4.4
10uM Orange Primer 1 Do not add to MM
10uM Blue Primer 1 Do not add to MM
Total 50 378.4
  • Add 43ul MM to each reaction
  • Incubate reactions at RT for 5 min
  • Put the tubes back on ice
  • Add Primers
  • Perform thermal cycling
Perform Kapa PCR Reaction
95C 3m 
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C

File:ZhangLab 2011-06-29 1.JPG

  1. A1-100 sperm cells
  2. A2-100 sperm cells (repeat)
  3. B1-100 ESCs
  4. B2-100 ESCs (repeat)
  5. C1-600pg GM12878 gDNA
  6. D-1.2ng GM12878 gDNA
  7. E1-NTC
  8. E2-NTC(MDA)

PAGE Analysis

File:ZhangLab 2 2011-06-29 16hr 00min.jpg

Results

  • E1-NTC was amplified and clear bands showed up on the gel image, high possibility of contamination
  • Possible source of contamination
  1. NTC water
  • E2-NTC added during MDA did not amplify during PCR that shows no contamination was introduced during MDA

To do

  • Size Selection on samples A1-E1 (use clone well gel to extract small amount of sample to do cloning)
  • Do cloning on size selected A1-E1 samples
  • Repeat experiment with only gDNA (UV EB buffer, use RT-PCR grade water as NTC)
  1. gDNA W proteinase k
  2. gDNA W/O proteinase k
  3. gDNA W protease
  4. NTC W proteinase K
  5. NTC W/O proteinase K
  6. gDNA W 0.1ul proteinase K
  • Run PCR on 5,8 and 9 with 3 sets of bisulfite verification primers
  • Pool samples A1-D
  • Perform 5 cycles of PCR to add illumina adapter and barcodes

E-Gel Size Select

  • UV EB
  1. Blank
  2. A1
  3. A2
  4. B1
  5. B2
  6. C1
  7. D
  8. E1
  • Ladder-1ul LM Ladder + 6ul EB
  • Samples- 10ul sample +10ul EB
  • Empty wells-30ul EB, add 10ul EB during run

File:ZhangLab 2011-06-29 3.jpg

TA Cloning

Ligation of PCR vector

Content Volume per Reaction MM
Fresh PCR Product 5.75 Do not add to MM
5X Ligation Buffer 2 15.4
pCR2.1 Vector (25ng/ul) 2 15.4
Sterile water 0 0
T4 DNA Ligase (4U/ul) 0.25 1.925
Total 10 77
  • Add 4.25ul MM to 5.75ul pcr product
  • Incubate the ligation reaction at 14°C overnight

6/30/11

Transformation

  • Equilibrate a water bath to 42°C.
  • Bring the S.O.C. medium to room temperature.
  • Spread each plate with 40 µl of 40 mg/ml X-Gal. Let the liquid soak into the plates.
  1. Thaw seven 50ul vial of One Shot® Competent Cells on ice.
  2. Add 2 μl ligation product into one vial of One Shot® Chemically Competent E. coli and mix by stirring gently with the pipette tip. Store the rest of the ligation products in -20 C. Do not mix by pipetting up and down.
  3. Incubate on ice for 30 minutes. Note: Longer incubations on ice do not seem to have any affect on transformation efficiency.
  4. Heat-shock the cells for 30 seconds at 42 °C without shaking.
  5. Immediately transfer the tubes to ice for 2 minutes.
  6. Add 250 μl of room temperature S.O.C. medium.
  7. Cap the tube tightly and shake the tube horizontally (256 rpm) at 37°C for 1 hour.
  8. Spread 20 μl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. (To ensure even spreading of small volumes, add 20 μl of S.O.C. medium. The manual recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.)

Sanger Sequencing